HESC-H9 and iPSC lines 3.5, 3.6 and 3.12 were analyzed using Affymetrix microarray before and after Definitive Endoderm (DE) formation. DE was induced using the ActivinA differentiation protocol described by D'Amour et al., 2006 (PMID: 16258519) Clustering analysis of transcripts that were differentially regulated during DE formation indicated that iPSC lines 3.5 and 3.12 differentiate in manner that is highly similar to HESC-H9 cells iPSC line 3.6 had a more divergent transcriptional profile.
Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.
No sample metadata fields
View SamplesGranulopoietic differentiation of myeloid progenitor cells derived from control iPSCs was performed in a two-step liquid culture. At the end of culture, stages of differentiation were identified by morphological analysis and submitted for RNA-sequencing analysis in order to provide insight into the genomic landscape of myeloid lineage hematopoiesis as modeled by the in vitro induced differentiation of iPSCs as compared to in vivo bone marrow-derived promyelocytes. Overall design: Peripheral blood from healthy controls was obtained and iPSC were generated from peripheral blood mononuclear cells. Hematopoietic progenitors generated from control iPSCs when cultured in myeloid expansion medium containing 50ng/mL SCF, 10ng/mL IL-3 and 10ng/mL GM-CSF for 5 days at which point cells were stained for CD45-Pacific blue, CD34-PECy7, CD33-AP, CD11b-APC-Cy7, CD15-FITC. 7-AAD was used to eliminate the dead cells. The promyelocytic population (CD45+CD34-CD33+CD11b-CD15+/lo) was sorted and the RNA from control iPSC promyelocytes was isolated using QIAGEN RNAeasy mini kit. The RNA samples were processed for RNA-seq analyses using RNA-seq protocol from NuGEN and Illumina. The amplified products were sequenced to analyze the gene expression profile of each replicate sample. A total of 20 samples were used in this analysis to characterize and compare iPSC in vitro differentiated myeloid cells with those isolated from human bone marrow.
p62 is required for stem cell/progenitor retention through inhibition of IKK/NF-κB/Ccl4 signaling at the bone marrow macrophage-osteoblast niche.
No sample metadata fields
View SamplesThe nuclear factor-kB (NF-kB) family of transcription factors is important for hematopoietic function, including development, maintenance, and differentiation of different hematopoietic lineages in response to cytokines and infection. Although ligand-independent or basal NF-kB signaling is required for HSC homeostasis in the absence of inflammation, the upstream tonic mediators of NF-kB signaling are not known. Herein we describe TNF receptor associated factor 6 (TRAF6) as an essential regulator of HSC homeostasis by preserving self-renewal and quiescence through basal activation of NF-kB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient HSPC revealed changes in adaptive immune signaling, innate immune signaling, and NF-kB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection occurs in HSPC and is required for HSC function. In addition, we established that loss of NF-kB signaling is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKb similarly resulted in impaired HSC self-renewal and fitness. Taken together, our observations position TRAF6 as an essential regulator of HSC homeostasis by maintaining a minimal threshold level of IKKb/NF-kB signaling. Overall design: Hematopoietic stem and progenitor cell (HSPC)-enriched lineage-Sca1+Kit+ (LSK) cells were sorted from mice reconstituted with Vav-Cre+ and Traf6-/-;Vav-Cre+ bone marrow cells. Total RNA was extracted from LSK cells and purified with Quick-RNA MiniPrep Kit (Zymogen). RNA quality was determined using the Agilent Bioanalyzer 2100 (Hewelett Packard). 100 ng total RNA was used for enrichment of poly A RNA with the Apollo 324 (WaferGen, Fremont, CA). Poly A RNA was further fragmented (RNase III), adaptor-ligated, and reverse transcribed (Superscript III reverse transcriptase, Lifetech, Grand Island, NY), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis IN). To prepare libraries, universal and index-specific primers, and sample-specific index were added to each adaptor-ligated cDNA sample to amplify the library, followed by purification using AMPure XP beads. The quality and yield of the libraries were assessed on the Bioanalyzer (Agilent, Santa Clara, CA). Libraries at the final concentration of 15 pM were clustered onto a PE flow cell using Illumina''s TruSeq PE Cluster kit v3, and sequenced using TruSeq SBS kit on the Illumina HiSeq system. To study differential gene expression, individually indexed libraries were proportionally pooled (20-50 million reads per sample in general) for clustering in cBot system (Illumina, San Diego, CA). Libraries at the final concentration of 15 pM were clustered onto a single read (SR) flow cell using Illumina's TruSeq SR Cluster kit v3, and sequenced for 50 bp using TruSeq SBS kit on Illumina HiSeq system.
TRAF6 Mediates Basal Activation of NF-κB Necessary for Hematopoietic Stem Cell Homeostasis.
Specimen part, Cell line, Subject
View SamplesMuscle biopsies taken from vastus lateralis muscle of 15 men and 15 women after 3 days of standardized diet and activity to examine effects of sex and age
Sex-related differences in gene expression in human skeletal muscle.
No sample metadata fields
View SamplesRNA from vastus lateralis of healthy young (21-31 year old) and older (62-77 year old) men. Signal data normalized to mean intensity of 500 over all probes sets. Analysis done with Affymetrix Microarray Suite 5.0 software.
Computational method for reducing variance with Affymetrix microarrays.
No sample metadata fields
View SamplesExostosin 1 (Ext1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of Ext1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos showed that Ext1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as Ext1 regulated genes. This analysis investigating the role of Ext1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development. Exostosin 1 (EXT1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of EXT1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. EXT1 MO embryos showed that EXT1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as EXT1 regulated genes. This analysis investigating the role of EXT1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development.
Zygotic expression of Exostosin1 (Ext1) is required for BMP signaling and establishment of dorsal-ventral pattern in Xenopus.
Treatment
View SamplesWe have generated a transgenic rat model with postnatal pathology. In order to investigate the potential contribution of changes in kidney gene expression to the pathology, we have conducted microarray-based gene expression profiling of postnatal kidney.
A novel long-range enhancer regulates postnatal expression of Zeb2: implications for Mowat-Wilson syndrome phenotypes.
Age, Specimen part, Time
View SamplesRNA from 5 mice with postdevelopmental knockout of myostatin and 5 mice with normal myostatin expression was analyzed with comprehensive oligonucleotide microarrays. Myostatin depletion affected the expression of several hundred genes at nominal P < 0.01, but fewer than a hundred effects were statistically significant according to a more stringent criterion (false discovery rate < 5%). Most of the effects were less than 1.5-fold in magnitude. In contrast to previously-reported effects of constitutive myostatin knockout, postdevelopmental knockout did not downregulate expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins involved in energy metabolism. Several collagen genes were expressed at lower levels in the myostatin-deficient muscles, and this led to reduced tissue collagen levels as reflected by hydroxyproline content. Myostatin knockout tended to down-regulate the expression of sets of genes with promoter motifs for Smad3, Smad4, myogenin, NF-B, serum response factor, and numerous other transcription factors. Main conclusions: in mature muscle, myostatin is a key transcriptional regulator of collagen genes, but not genes encoding contractile proteins or genes encoding proteins involved in energy metabolism.
Skeletal muscle gene expression after myostatin knockout in mature mice.
Sex, Age, Specimen part
View SamplesMuscle biopsies taken from vastus lateralis muscle of 30 normal subjects and 19 FSHD subjects (see PubMed ID 17151338)
Expression profile of FSHD supports a link between retinal vasculopathy and muscular dystrophy.
No sample metadata fields
View SamplesHepatocytes are polarized epithelial cells whose function depends upon their ability to distinguish between the apical and basolateral surfaces that are located at intercellular tight junctions. It has been proposed that the signaling cascades that originate at these junctions influence cellular activity by controlling gene expression in the cell nucleus. To assess the validity of this proposal with regard to hepatocytes, we depleted expression of the tight junction protein junctional adhesion molecule-A (JAM-A) in the HepG2 human hepatocellular carcinoma cell line. Reduction of JAM-A resulted in a striking change in cell morphology, with cells forming single-layered sheets instead of the normal multi-layered clusters. In the absence of JAM-A, other tight junction proteins were mislocalized, and canaliculi, which form the apical face of the hepatocyte, were consequently absent. While most changes in gene expression were modest, there was a strong transcriptional induction of the adherens junction protein E-cadherin in cells with reduced levels of JAM-A. This increase in E-cadherin was partially responsible for the observed alterations in cell morphology and mislocalization of tight junction proteins. We therefore propose that we have uncovered a novel mechanism for crosstalk between specific components of tight and adherens junctions that can be utilized to regulate adhesion between hepatic cells and to maintain hepatocyte cell polarity.
Junctional adhesion molecule-A is critical for the formation of pseudocanaliculi and modulates E-cadherin expression in hepatic cells.
No sample metadata fields
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